<p>Cytochrome <i>cd</i>₁ nitrite reductase is a bifunctional enzyme, which catalyses the one-electron reduction of nitrite to nitric oxide, and the four-electron reduction of oxygen to water. The latter is a cytochrome oxidase reaction. Both reactions occur on the <i>d</i>₁ haem iron of the enzyme.</p><p>Time resolved crystallographic studies presented here show that the mechanisms of nitrite and oxygen reduction share common elements. This is of interest from an evolutionary point of view since aerobic respiratory enzymes are thought to have evolved from denitrifying enzymes. Despite of similarities, the results also imply different requirements for the timing of electron transfer to the active site in these reactions.</p><p>Quantum chemical calculations suggest that nitric oxide, the product of nitrite reduction, is not spontaneously released from the haem iron while this is not the case with water. Reduction of the haem while nitric oxide is still bound to it would result in a tight dead-end complex. A mechanism must therefore exist for the selective control of electron transfer during the reaction.</p><p>Structural studies with a product analogue (carbon monoxide) combined with flash photolysis of the complex in solution revealed an unexpected proton uptake by the active site as the neutral CO molecule left the enzyme. This led to the suggestion that the increased positive potential of the active site triggers preferential electron transfer when the active site is empty.</p><p>Crystallisation and structure determination of the reduced enzyme revealed extremely large domain rearrangements. These results offer insights into the role of tethered electron shuttle proteins in complex redox systems, and suggests a mechanism for conformational gating in catalysis.</p>

Cytochrome cd1 nitrite reductase is a bifunctional enzyme, which catalyses the one-electron reduction of nitrite to nitric oxide, and the four-electron reduction of oxygen to water. The latter is a cytochrome oxidase reaction. Both reactions occur on the d1 haem iron of the enzyme. Time resolved crystallographic studies presented here show that the mechanisms of nitrite and oxygen reduction share common elements. This is of interest from an evolutionary point of view since aerobic respiratory enzymes are thought to have evolved from denitrifying enzymes. Despite of similarities, the results also imply different requirements for the timing of electron transfer to the active site in these reactions. Quantum chemical calculations suggest that nitric oxide, the product of nitrite reduction, is not spontaneously released from the haem iron while this is not the case with water. Reduction of the haem while nitric oxide is still bound to it would result in a tight dead-end complex. A mechanism must therefore exist for the selective control of electron transfer during the reaction. Structural studies with a product analogue (carbon monoxide) combined with flash photolysis of the complex in solution revealed an unexpected proton uptake by the active site as the neutral CO molecule left the enzyme. This led to the suggestion that the increased positive potential of the active site triggers preferential electron transfer when the active site is empty. Crystallisation and structure determination of the reduced enzyme revealed extremely large domain rearrangements. These results offer insights into the role of tethered electron shuttle proteins in complex redox systems, and suggests a mechanism for conformational gating in catalysis.